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Copyright © 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case
Chapter 6
Microbial Growth
© 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case
© 2013 Pearson Education, Inc.
© 2013 Pearson Education, Inc.
Learning Objectives
The Requirements for Growth
6-1 Classify microbes into five groups on the basis of preferred temperature range.
6-2 Identify how and why the pH of culture media is controlled.
6-3 Explain the importance of osmotic pressure to microbial growth.
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Microbial Growth
Increase in number of cells, not cell size Populations Colonies
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The Requirements for Growth
Physical requirements Temperature pH Osmotic pressure
Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor
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Psychrotrophs
Grow between 0°C and 20–30°C Cause food spoilage
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Figure 6.2 Food preservation temperatures.
Temperatures in this range destroy most microbes, although lower temperatures take more time.
Very slow bacterial growth.
Rapid growth of bacteria; some may produce toxins.
Many bacteria survive; some may grow. Refrigerator temperatures; may allow slow growth of spoilage bacteria, very few pathogens. No significant growth below freezing.
Danger zone
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Figure 6.3 The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage.
Refrigerator air
5 cm (2′′) deep
15 cm (6′′) deep
Approximate temperature range at which Bacillus cereus multiplies in rice
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pH
Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Acidophiles grow in acidic environments
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Chemical Requirements
Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2
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Chemical Requirements
Nitrogen In amino acids and proteins Most bacteria decompose proteins Some bacteria use NH4
+ or NO3–
A few bacteria use N2 in nitrogen fixation
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Chemical Requirements
Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors
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Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria
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Singlet oxygen: 1O2− boosted to a higher-energy
state Superoxide free radicals: O2
Peroxide anion: O22–
Hydroxyl radical (OH•)
Toxic Oxygen
Superoxide dismutase O2 + O2 + 2 H+ H2O2 + O2
Catalase 2 H2O2 2 H2O + O2
Peroxidase H2O2 + 2 H+ 2 H2O
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Organic Growth Factors
Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines
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Learning Objectives
Biofilms
6-7 Describe the formation of biofilms and their potential for causing infection.
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Biofilms
Microbial communities Form slime or hydrogels Bacteria attracted by chemicals via quorum sensing
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Figure 6.5 Biofilms.
Clumps of bacteria adhering to surface
Surface Water currents
Migrating clump of bacteria
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Biofilms
Share nutrients Sheltered from harmful factors
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Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.
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Biofilms
Patients with indwelling catheters received contaminated heparin
Bacterial numbers in contaminated heparin were too low to cause infection
84–421 days after exposure, patients developed infections
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Culture Media
Culture medium: nutrients prepared for microbial growth
Sterile: no living microbes Inoculum: introduction of microbes into medium Culture: microbes growing in/on culture medium
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Agar
Complex polysaccharide Used as solidifying agent for culture media in Petri
plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies at ~40°C
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Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli
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Anaerobic Culture Methods
Reducing media Contain chemicals (thioglycolate or oxyrase) that
combine O2 Heated to drive off O2
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BSL-1: no special precautions BSL-2: lab coat, gloves, eye protection BSL-3: biosafety cabinets to prevent airborne
transmission BSL-4: sealed, negative pressure Exhaust air is filtered twice
Biosafety Levels
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Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.
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Make it easy to distinguish colonies of different microbes
Differential Media
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Enrichment Culture
Encourages growth of desired microbe Assume a soil sample contains a few
phenol-degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil,
and incubate Transfer 1 ml to another flask of the phenol medium, and
incubate Transfer 1 ml to another flask of the phenol medium, and
incubate Only phenol-metabolizing bacteria will be growing
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Learning Objectives
Obtaining Pure Cultures
6-11 Define colony. 6-12 Describe how pure cultures can be isolated by
using the streak plate method.
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Obtaining Pure Cultures
A pure culture contains only one species or strain A colony is a population of cells arising from a
single cell or spore or from a group of attached cells A colony is often called a colony-forming unit
(CFU) The streak plate method is used to isolate pure
cultures
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Figure 6.11 The streak plate method for isolating pure bacterial cultures.
Colonies
1
2
3
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Learning Objectives
Preserving Bacterial Cultures
6-13 Explain how microorganisms are preserved by deep-freezing and lyophilization (freeze-drying).
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Preserving Bacterial Cultures
Deep-freezing: –50° to –95°C Lyophilization (freeze-drying): frozen
(–54° to –72°C) and dehydrated in a vacuum
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If 100 cells growing for 5 hours produced 1,720,320 cells:
ANIMATION Binary Fission
Generation Time
Number of generations = Log number of cells (end) − Log number of cells (beginning)
0.301
Generation time = 60 min × hours
Number of generations = 21 minutes/generation
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Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed line) and arithmetically (solid line).
Log10 = 1.51
Log10 = 3.01
Log10 = 4.52
Log10 = 6.02 (1,048,576)
Generations
Log 1
0 of
num
ber o
f cel
ls
Num
ber o
f cel
ls
(32) (1024) (32,768)
(65,536) (131,072)
(262,144)
(524,288)
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ANIMATION Bacterial Growth Curve
Phases of Growth
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Lag Phase Intense activity preparing for population growth, but no increase in population.
Log Phase Logarithmic, or exponential, increase in population.
Stationary Phase Period of equilibrium; microbial deaths balance production of new cells.
Death Phase Population Is decreasing at a logarithmic rate.
The logarithmic growth in the log phase is due to reproduction by binary fission (bacteria) or mitosis (yeast).
Figure 6.15 Understanding the Bacterial Growth Curve.
Staphylococcus spp.
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Figure 6.16 Serial dilutions and plate counts.
Original inoculum
1 ml 1 ml 1 ml 1 ml 1 ml
9 m broth in each tube
Dilutions 1:10 1:100 1:1000 1:10,000 1:100,000
1 ml 1 ml 1 ml 1 ml 1 ml
1:10 1:100 1:1000 1:10,000 1:100,000 (10-1) (10-2) (10-3) (10-4) (10-5)
Plating
Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml (For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)
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After incubation, count colonies on plates that have 25–250 colonies (CFUs)
Plate Counts
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Figure 6.17 Methods of preparing plates for plate counts.
The pour plate method The spread plate method
Inoculate empty plate.
Add melted nutrient agar.
Swirl to mix.
Colonies grow on and in solidified medium.
1.0 or 0.1 ml 0.1 ml
Bacterial dilution
Inoculate plate containing solid medium.
Spread inoculum over surface evenly.
Colonies grow only on surface of medium.
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Figure 6.18 Counting bacteria by filtration.
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Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.
Grid with 25 large squares
Cover glass
Slide
Bacterial suspension is added here and fills the shallow volume over the squares by capillary action.
Bacterial suspension Cover glass
Slide
Cross section of a cell counter. The depth under the cover glass and the area of the squares are known, so the volume of the bacterial suspension over the squares can be calculated (depth × area).
Microscopic count: All cells in several large squares are counted, and the numbers are averaged. The large square shown here has 14 bacterial cells.
The volume of fluid over the large square is 1/1,250,000 of a milliliter. If it contains 14 cells, as shown here, then there are 14 × 1,250,000 = 17,500,000 cells in a milliliter.
Location of squares
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Direct Microscopic Count
Number of bacteria/ml = Number of cells counted Volume of area counted
14 8 × 10−7 = 17,500,000
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Figure 6.21 Turbidity estimation of bacterial numbers.
Light source
Light
Blank
Spectrophotometer
Light-sensitive detector Scattered light
that does not reach detector
Bacterial suspension
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Measuring Microbial Growth
Direct Methods Plate counts Filtration MPN Direct microscopic count
Indirect Methods Turbidity Metabolic activity Dry weight